Applications and Limitations of Rapid Methods

Almost all rapid methods are designed to detect a single target, which makes them ideal for use in quality control programs to quickly screen large numbers of food samples for the presence of a particular pathogen or toxin. A positive result by a rapid method however, is only regarded as presumptive and must be confirmed by standard methods. Although confirmation may extend analysis by several days, this may not be an imposing limitation, as negative results are most often encountered in food analysis.

Most rapid methods can be done in a few minutes to a few hours, so they are more rapid than traditional methods. But, in food analysis, rapid methods still lack sufficient sensitivity and specificity for direct testing; hence, foods still need to be culture-enriched before analysis. Although enrichment is a limitation in terms of assay speed, it provides essential benefits, such as diluting the effects of inhibitors, allowing the differentiation of viable from non-viable cells and allowing for repair of cell stress or injury that may have resulted during food processing.

Evaluations of rapid methods show that some perform better in some foods than others. This can be attributed mostly to interference by food components, some of which can be especially troublesome for the technologies used in rapid methods. For example, an ingredient can inhibit DNA hybridization or Taq polymerase, but has no effect on antigen-antibody interactions and the converse situation may also occur. Since method efficiencies may be food dependent, it is advisable to perform comparative studies to ensure that a particular assay will be effective in the analysis of that food type.

The specificity of DNA based assays is dictated by short probes; hence, a positive result, for instance with a probe or primers specific for a toxin gene, only indicates that bacteria with those gene sequences are present and that they have the potential to be toxigenic. But, it does not indicate that the gene is actually expressed and that the toxin is made. Likewise, in clostridial and staphylococcal intoxication, DNA probes and PCR can detect only the presence of cells, but are of limited use in detecting the presence of preformed toxins.
Currently, there are at least 30 assays each for testing for E. coli O157:H7 and for Salmonella. Such a large number of options can be confusing and overwhelming to the user, but, more importantly, has limited the effective evaluation of these methods. As a result, only few methods have been officially validated for use in food testing .

Reference:
Feng, P. 1997. Impact of Molecular Biology on the Detection of Foodborne Pathogens. Mol. Biotech. 7:267-278.

Bacillus Cereus Diarrhetic Enterotoxins

Bacillus cereus is an aerobic spore former that is commonly found in soil, on vegetables, and in many raw and processed foods. Consumption of foods that contain large numbers of B. cereus (106 or more/g) may result in food poisoning. Although certain physiological and cultural characteristics are necessary for identifying B. cereus, its enterotoxigenicity indicates whether a suspect strain may be a public health hazard.

Common procedure to detect Diarrhetic Enterotoxins of Bacillus Cereus is by two methods. This is by using a semisolid agar medium and a serological procedure (the microslide gel double diffusion test) to identify the enterotoxin.

Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 15. Bacillus cereus Diarrheal Enterotoxin
*Author: Reginald W. Bennett

Main Principles of ELISA

Immunoenzymatic test

Bacteris Toxins

Staphylococcal Enterotoxins Detection and Identification

When large numbers of enterotoxigenic staphylococci grow in foods, they may elaborate enough toxin to cause food poisoning after the foods are ingested. The most common symptoms of staphylococcal food poisoning, which usually begin 2-6 h after contaminated food is consumed, are nausea, vomiting, acute prostration, and abdominal cramps. To date, 8 enterotoxins (types A, B, C1, C2, C3, D, E, and H) have been identified as distinct serological entities. Current methods to detect enterotoxins use specific polyclonal or monoclonal antibodies (33,42,43).

Detection Methods and Techniques
1. Microslide gel double diffusion technique
2. Radioimmunoassay agglutination (RIA)
3. Enzyme-linked immunosorbent assay (ELISA)
- "double antibody sandwich" ELISA is the method of choice, because reagents are commercially available in polyvalent and monovalent formats for both toxin screening and serotype specific identification
- An automated enzyme-linked fluorescent immunoassay (ELFA) has been developed and is commercially available. This method has undergone specificity and sensitivity evaluations and has proven to be an effective serological system for the identification of staphylococcal enterotoxin in a wide variety of foods
4. Other methods, which have been used in the identification of the staphylococcal enterotoxins and may have application in foods, are the T-cell proliferation assay, and polyacrylamide gel electrophoresis (PAGE) combined with Western blotting

Summary

Microslide gel double diffusion precipitation test, two manual ELISAs [TecraTM, TransiaTM], an automated qualitative "enzyme-linked fluorescent immunoassay" [ELFATM, VidasTM], and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]-immunoblotting) for the identification of staphylococcal enterotoxin from isolates and from foods. - most common and often practiced and used


Recommended for routine analysis of foods for staphylococcal enterotoxin is the use, initially, of two different polyvalent ELISA kits



Known limits that causes food borne illness

When large numbers of enterotoxigenic staphylococci grow in foods, they may elaborate enough toxin to cause food poisoning after the foods are ingested. The most common symptoms of staphylococcal food poisoning, which usually begin 2-6 h after contaminated food is consumed, are nausea, vomiting, acute prostration, and abdominal cramps. To date, 8 enterotoxins (types A, B, C1, C2, C3, D, E, and H) have been identified as distinct serological entities. Current methods to detect enterotoxins use specific polyclonal or monoclonal antibodies (33,42,43).

Detection of DSP and PSP

For routine monitoring of DSP toxins, a mouse bioassay procedure developed by Yasumoto et al. is widely used. Although individual methods vary in detail, all assays invlove the extraction of lipids-soluble DSP toxins with acetone, removal of the solvent and injection of the extract into a pre-weighed mouse . To confirm positiveresults obtained using the bioassay test, it is standard practivce to carry out an independent analysis by HPLC using 9-anthryldiazomethane derivatives of the DSP toxins. HPLC can detect of 15 micrograms or less of okadaic acid per 100g whole tissue. As for PSP toxins, it is recognised that there is an urgent need to develop alternative methods to the mouse bioassay which do not rely on the use of live animals

Adapted from Naturally Occuring Toxins, 42nd report on Chemical Aspects of Food Survelliance, 1994, MAFF

DSP...Diarrhetic Shell Fish Toxins

DSP toxins, so named because of their ability to induce gastrointestinal disorders in humans, are a group of 1 polyether carboxylic acids. The first DSP toxin to be identified was dinophysis toxin, then Okadaic acid from blue mussels and dinophysis toxin-3, a mixture of unsaturated fatty acid esters of DTX-1, has been isolated from the digestive gland of scallop.

Symptoms: mian cause diarrhea

the food Safety (Live Bivalve Molluscs) REgulations 1993, which incooperates into national law (UK) the requirements of EC Directive 91/492/EEC. Under the regulations, 'the customary biological methods must not give a positive result to the presence of DSP in the edible parts of molluscs'. - which means 0 or absent always

Aqautic Toxins...PSP

This is some and part of mine research...

Paralytic shellfish poisoning toxins (PSP)....very important

The toxins in shellfish most commonly associatiated with PSP consist of around 20 closely related compounds. These toxins are of three types: Carbomate; N-sulphocarbamoyl; and decarbomoyl toxins.

Nature and severity of PSP toxins vary with the quantity of toxins ingested and proportions of the individual toxins involved. Effects of PSP toxins on humans are well established. the most common symptoms following ingestion are paraesthesia of the perioral region, tongue and limbs, with ataxia, lower back pain and a sensation of consciousness and respiratory failure can occur whcih may be fatal in the absence of respiratory support.

Standard acceptale limit in regulations is 80microgram/100g
The maximum permitted limit of PSP toxins in bivalve molluscs has been subject to specific legislation in the form of The FOos Safety (LIve Bivelve Molluscs) Regulation 1992 (S.I. [1992] No. 3164). under the regulations, ' the total concentration of PSP toxins in the edible parts of molluscs must not exceed 80microgram/100g of molluscs flesh'.

Analysis of PSP toxins can be through the mouse bioassay method

Controversy of GM foods....

THIS IS ALSO A SUMMARY OF THE CONTROVERSY.....WE WILL BUILD ON THIS POINTS....

Controversies


Safety

Potential human health impact: allergens, transfer of antibiotic resistance markers, unknown effects Potential environmental impact: unintended transfer of transgenes through cross-pollination, unknown effects on other organisms (e.g., soil microbes), and loss of flora and fauna biodiversity

Access and Intellectual Property

Domination of world food production by a few companies
Increasing dependence on Industralized nations by developing countries
Biopiracy—foreign exploitation of natural resources

Ethics

Violation of natural organisms' intrinsic values
Tampering with nature by mixing genes among species
Objections to consuming animal genes in plants and vice versa
Stress for animal

Labeling

Not mandatory in some countries (e.g., United States)
Mixing GM crops with non-GM confounds labeling attempts

Society

New advances may be skewed to interests of rich countries

Reference Site:
http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml

Benefits of GM food....

THIS IS A SUMMARY OF WAD I HAVE FOUND FROM MINE RESEARCH..THINK WE CA DO FURTHER RESEARCH ON EACH OF THIS POINTS

Benefits

Crops

Enhanced taste and quality
Reduced maturation time
Increased nutrients, yields, and stress tolerance
Improved resistance to disease, pests, and herbicides
New products and growing techniques

Animals

Increased resistance, productivity, hardiness, and feed efficiency
Better yields of meat, eggs, and milk
Improved animal health and diagnostic methods

Environment

"Friendly" bioherbicides and bioinsecticides
Conservation of soil, water, and energy
Bioprocessing for forestry products
Better natural waste management
More efficient processing

Society

Increased food security for growing populations

Reference Site

http://www.ornl.gov/sci/techresources/Human_Genome/elsi/gmfood.shtml

About overseas local authority.....FSIS

Adapted from http://www.fsis.usda.gov/About_FSIS/index.asp

The Food Safety and Inspection Service (FSIS) is the public health agency in the U.S. Department of Agriculture responsible for ensuring that the nation's commercial supply of meat, poultry, and egg products is safe, wholesome, and correctly labeled and packaged.

....THE OFFICIAL ROLE OF THIS AGENCY....KNOWN TO BE WITHIN THE USDA.....


more will be from evelyn....